The blood level of thioredoxin 1 as a supporting biomarker in the detection of breast cancer

Background There is a long-time unmet need for a means to detect breast cancer (BC) using blood. Although mammography is accepted as the gold standard for screening, a blood-based diagnostic can complement mammography and assist in the accurate detection of BC in the diagnostic process period of early diagnosis. We have previously reported the possible use of thioredoxin 1 (Trx1) in serum as a novel means to detect BC. In the present study, we validated the clinical utility of Trx1 to identify BC by testing sera from biopsy-confirmed cancer patients and women without cancer. Methods We have generated monoclonal antibodies against Trx1 and developed an ELISA kit that can quantitate Trx1 in sera. The level of Trx1 was determined in each serum from women without cancer (n = 114), as well as in serum from patients with BC (n = 106) and other types of cancers (n = 74), including cervical, lung, stomach, colorectal, and thyroid cancer. The sera from BC patients were collected and classified by the subjects’ age and cancer stage. In addition to the Trx1 levels of BC patients, several pathological and molecular aspects of BC were analyzed. Test results were retrospectively compared to those from mammography. Each test was duplicated, and test results were analyzed by ROC analysis, one-way ANOVA tests, and unpaired t-tests. Results The mean level of Trx1 from women without cancer was 5.45 ± 4.16 (±SD) ng/ml, that of the other malignant cancer patient group was 2.70 ± 2.01 ng/ml, and that from the BC group was 21.96 ± 6.79 ng/ml. The difference among these values was large enough to distinguish BC sera from non-BC control sera with a sensitivity of 97.17% and specificity of 94.15% (AUC 0.990, p < 0.0001). Most Trx1 levels from BC patients’ sera were higher than the cut-off value of 11.4 ng/ml regardless of age, stage, histological grade, type, and specific receptors’ expression profile of BC. The level of Trx1 could rescue women from most cases of misread or incomplete mammography diagnoses. Conclusion These results indicated that the blood level of Trx1 could be an effective and accurate means to assist the detection of BC during the early diagnosis period.

Detection of Selenium/Sulphur substitution in the heterologous expression of Thioredoxin isoform 1 (Trx1) from Saccharomyces cerevisiae.

Thioredoxins are ubiquous proteins with 2 cysteines at the active site. The isoform 1 of Thioredoxin from Saccharomyces cerevisae (Trx1) has six sulphur aminoacids, two cysteines and four methionines. In this work we performed the replacement of cysteines by selenocysteines by growth of a transformed celular expression vector E. coli BL21-DE3 in selenocysteine containing culture medium. The Maldi-TOF spectra of Seleno/Sulphur substituted Trx1 revealed six component peaks with 46-48 Da range between them, that is the isotopic Seleno-Sulfur difference, showing the replacement of the Cysteines and Methionines to Selenocysteines and Selenomethionines. The Maldi-TOF spectra of the peptides derived from Trypsin digestion of the purified Thioredoxin (peptide mass fingerprint) show Selenocysteine and Selenomethionine containing peptides. Therefore we are demonstrating that cystein can be replaced by selenocystein and be metabolically converted to selenomethionine during Trx1 heterologous translation. Furthermore, the Maldi-TOF spectra are showing the presence of the most abundant isotopes of selenium inserted in the peptides containing cysteine and methionine, derived from the Trx1 digestion. The one dimensional 77 Se − 1 H heteronuclear multiple quantum coherence NMR spectroscopy (1D-HMQC) for reduced Seleno substituted Trx1 (Se-Trx1), revealed three ressonance lines for 1 H β 1 from Selenocysteines 30 and 33, between 1.6 and 2,0 ppm. The bidimensional HMQC spectra (2D-HMQC) of the reduced Se Trx1 show the 77 Se ressonance signal in 178 ppm, coupled with 1 H β 1 and 1 H β 2 lines between 2.1 and 1.8 ppm. The 1D-HMQC for oxidized Trx1 revealed the only one broad resonance in 2.6 ppm probably relative to the 1 H β 1 protons. The 2D-HMQC spectrum of oxidized protein shows a higher chemical shift of selenocysteine 77 Se (832 ppm) if compared to reduced state (178 ppm). Together these data are showing that the protocol of Se − S substitution developed here is a efficient method to label the active site of Thioredoxin 1 with a broad band chemical shift atom 77 Se. Furthermore the large spectral window of the 77 Se NMR detected between reduced and oxidized states of the Thioredoxin 1 shows that this atom is an excellent probe for accessing oxidative states and probably the conformational dynamics of the active site of the Se-Trx1.

Oxidized thioredoxin-1 restrains the NLRP1 inflammasome

At least six human proteins detect danger-associated signals, assemble into complexes called inflammasomes, and trigger pyroptotic cell death. NLRP1 was the first protein discovered to form an inflammasome, but the danger signals and molecular mechanisms that control its activation have not yet been fully established. Here, we report that the NACHT-LRR region of NLRP1 directly binds to oxidized form of thioredoxin-1 (TRX1). We found that NLRP1 requires the ATPase activity of its NACHT domain to associate with TRX1, and that this interaction represses inflammasome activation. Moreover, we discovered that several patient-derived missense mutations in the NACHT-LRR region of NLRP1 weaken TRX1 binding, leading to inflammasome hyperactivation and autoinflammatory disease. Overall, our results establish that oxidized TRX1 binds to and restrains the NLRP1 inflammasome, thereby revealing a link between the cellular redox environment and innate immunity.